Objective: To investigate the effects of injured airway epithelial cells on the transdifferentiation of sub-epithelial fibroblasts, thus addressing the mechanisms of characteristic airway remodeling in asthmatic airway.
Methods: Human sub-epithelial fibroblasts (SEF) or SEF in collagen gels were co-cultured with culture dishes which containing or not containing bronchial epithelial cells (16HBE) for 4 days. The experiments were divided into 7 groups according to different treatment to 16HBE; Group A: culture dishes not containing 16HBE; Group B: culture dishes containing normal 16HBE; Group C: culture dishes containing 16HBE treated with mechanical scrape; Group D: culture dishes containing 16HBE treated with mechanical scrape plus LPS stimulation; Group E: culture dishes containing 16HBE pre-added with endothelin receptor A inhibitor (BQ123) followed by treatment with mechanical scrape plus LPS stimulation; Group F: culture dishes containing 16HBE pre-added with TGF-beta(1) neutralizing antibody followed by treatment with mechanical scrape plus LPS stimulation; Group G: culture dishes containing 16HBE pre-added with BQ123 plus TGF-beta(1) neutralizing antibody followed by treatment with mechanical scrape plus LPS stimulation. The expression of alpha-smooth muscle actin (alpha-SMA) in the SEF was assessed by immunofluorescence or Western blotting. The diameters of the collagen gels containing SEF (8 samples in each group) were measured. Moreover, SEF in group A, B or D were selected, and the dynamic changes of intracellular calcium concentration [Ca(2+)]i were observed (at least 6 cells were analyzed in each group) by laser co-focal microscopy in the transdifferentiated SEF in response to the stimulation of the supernatant of 16HBE treated with mechanical scrape plus LPS stimulation for 24 h.
Results: alpha-SMA expression in SEF was most abundant in group D compared with other groups. Relative contraction percentage was also significantly higher in group D [(53 +/- 8)%] than that in group B [(10 +/- 10)%], group C [(24 +/- 8)%], group E [(36 +/- 9)%], group F [(37 +/- 3)%] or group G [(31 +/- 7)%], respectively (F = 24.80, 38.10, all P < 0.01). Increased fluorescence intensity of [Ca(2+)]i was observed in the SEF stimulated with the supernatant of the injured 16HBE in all groups, which was significant in group D (F = 16.60, 8.97; all P < 0.01).
Conclusions: Injured airway epithelial cells induced the transdifferentiation of SEF towards alpha-SMA-expressing and elevated contractile myofibroblasts, in which both ET-1 and TGF-beta(1) may play crucial roles. The supernatant of injured airway epithelial cells-induced influx of [Ca(2+)]i may be an early signal for initiating the increased contractility in the transdifferentiated SEF.